Impairment of adult hippocampal neural progenitor proliferation by methamphetamine: role for nitrotyrosination

Abstract

Methamphetamine (METH) abuse has reached epidemic proportions, and it has become increasingly recognized that abusers suffer from a wide range of neurocognitive deficits. Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs). It is now well established that new neurons are continuously generated from NPCs in the adult hippocampus, and accumulating evidence suggests important roles for these neurons in hippocampal-dependent cognitive functions. In a rat hippocampal NPC culture system, we find that METH results in a dose-dependent reduction of NPC proliferation, and higher concentrations of METH impair NPC survival. NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH. We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress. Further, we identify seventeen NPC proteins that are post-translationally modified via 3-nitrotyrosination in response to METH, using mass spectrometric approaches. One such protein was pyruvate kinase isoform M2 (PKM2), an important mediator of cellular energetics and proliferation. We identify sites of PKM2 that undergo nitrotyrosination, and demonstrate that nitration of the protein impairs its activity. Thus, METH abuse may result in impaired adult hippocampal neurogenesis, and effects on NPCs may be mediated by protein nitration. Our study has implications for the development of novel therapeutic approaches for METH-abusing individuals with neurologic dysfunction and may be applicable to other neurodegenerative diseases in which hippocampal neurogenesis is impaired.

Figure 1

Establishment of NPC culture system in vitro. Adult hippocampal progenitors (NPCs) derived from Fisher rats were maintained under either proliferative or differentiating conditions. A. Under proliferative conditions, over 99% of cells are co-labeled with antibodies to nestin (green, cytoplasmic) and Ki67 (red, nuclear), markers of proliferating cells. B,C. Cells are exposed to proliferative conditions for 24 hrs in the presence of BrdU, followed by 5 days of differentiation conditions stain for BrdU (green), indicating that they were all initially proliferating cells. In addition, some cells in B co-label with Tuj1 (red, neuron), or RIP (blue, oligodendrocyte) and in C co-label with GFAP (blue, astrocyte). D. Immunostaining of cells derived from clonal NPCs grown under proliferative (FGF-2) or differentiating (FBS+RA) conditions yields reproducible percentages of Tuj1 (neuronal), GFAP (astrocytic), and RIP (oligodendroglial) positive cells.

Figure 2

METH decreases BrdU incorporation and causes apoptosis in vitro. NPCs maintained under proliferative conditions were exposed to various concentrations of METH for 48 hours. BrdU (5 uM) was added for 4 hours to label proliferating cells. Cells were immunostained with antibody against BrdU and counterstained with DAPI. A. Quantitation of BrdU positive cells reveals a dose- and time-dependent decrease in BrdU positive cells upon exposure to METH (numbers following METH refer to concentration in µM). * p <0.01, ** p <0.001 ANOVA compared to control (proliferation conditions). B. Cells were stained with Trypan Blue and visualized under light microscopy. Numbers following M refer to concentration of METH in µM. * p <0.01, ** p <0.001. C. TUNEL positive cells were not detected in control cells, but were seen in some cells exposed for 48 hrs to METH 1000 µM (white arrows).

Figure 3

METH does not affect differentiation of NPCs. A,B. NPCs maintained under proliferative conditions were exposed to METH (250 uM) for 24 hours, and fixed and immunostained 4 days later with antibody against Tuj1, GFAP, and RIP, and counterstained with DAPI. METH treatment does not result in premature differentiation (A), but does result in fewer total numbers of differentiated cells (B). C,D. NPCs in proliferating media were exposed to METH for 24 hrs, followed by culture for 4 days in differentiating conditions. METH does not alter the percentage of cells adopting neuronal, astrocytic, or oligodendrocyte markers. E, F. NPCs were cultured in differentiating conditions for 24 hours, followed by METH treatment for an additional 24 hours. Cells were analyzed 4 days after being placed in differentiating conditions. METH treatment after the onset of differentiation does not significantly affect fate choice (E) or numbers of differentiating cells (F). * p <0.01 ANOVA, compared to control.

Figure 4

METH induces oxidative stress in NPCs. NPCs maintained under proliferative conditions were exposed to various concentrations of METH. Numbers following "M" refer to concentration of METH in uM. 3-NP, DMNQ, and STS are inducers of oxidative stress and were used as positive controls. A. NPCs exposed to METH for 4 hrs were loaded with Carboxy-H2 DCFDA. ROS formation (green) is far greater in cells exposed to METH (250 and 500 uM) than control. B. Nitrite production from supernatants of NPCs was assessed via the Griess reaction. Values were normalized to supernatants from untreated NPCs.C. 3-nitrotyrosination of cell lysates increases in a dose-dependent manner in NPCs exposed to METH as demonstrated by quantification of slot blots, suggesting increased oxidative and/or nitrosative stress. D. Lysates from untreated NPCs (Control) or those exposed to STS, METH 250 uM (M250) and 1000 uM (M1000) for 24 hours were subjected to western blotting with 3-nitrotyrosine antibody. Several bands, including those of apparent molecular mass 10, 25, 30, 32, 45, and 90 kDa, were preferentially nitrotyrosinated in NPCs exposed to METH (250 and 1000 uM) as compared to control cells. * p <0.05, ** p <0.01 ANOVA.

Figure 5

Effects of antioxidants on METH-induced cell death of NPCs. NPCs were preincubated for 2 hrs with either Trolox (10 uM) or uric acid (UA, 25 or 250 uM), followed by addition of METH (250 uM). Cells were analyzed at 48 hours. (A) Protein lysates were subjected to Western blotting with antibody against 3-nitrotyrosine. Both Trolox and Uric acid markedly decrease nitrotyrosination of a number of NPC proteins. (B). Both Trolox and Uric acid protect against METH-induced NPC death and decrease reactive oxygen species-induced nitrotyrosination (** p <0.01, ANOVA).

Figure 6

METH induces nitrotyrosination of PKM2 and inhibits its activity. A. NPC lysates were immunoprecipitated with antibody to 3-nitrotyrosine, and ratio of nitrotyrosinated to total PKM2 is quantified. B. Assessment of pyruvate kinase activity in NPC lysates. C. 3-nitrotyrosine Western blotting (above) and pyruvate kinase activity (below) of recombinant PKM2 (control or peroxynitrite treated). *p <0.05, ***p <0.001 ANOVA.

Figure 7

Modeling of the nitrated tyrosine residues of PKM2. A) Location of the nitrated tyrosines on one subunit of the PK tetramer. Nitrated tyrosine residues are represented as sticks colored by atom type (green, carbon; blue, nitrogen; red, oxygen). Detailed view of the environment of the nitrated tyrosines. B) Tyr 175 at the interface of the A-domain (green) and B-domain of neighbor subunit (magenta). C) Tyr 105 at the B/C interface. Possible and actual hydrogen bonds are represented as dashed blue lines. Steric contacts are indicated with dashed red lines. Modeling and figures were done with the program PyMOL (TM Schrodinger, LLC).